A Novel Pathway of Platelet Activation in ACS Mediated by LL-37 Immunoglobulin G Autoantibody Immune Complexes

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SUMMARY
The cathelicidin antimicrobial peptide LL-37 is a self-antigen in neutrophil extracellular traps that provokes autoantibody responses in autoimmune/autoinflammatory conditions.LL-37 immunoglobulin (Ig) G autoantibody levels were measured in subjects with and without atherosclerotic cardiovascular disease assessed using the coronary artery calcium score, in patients who had a future myocardial infarction and in a cohort of acute coronary syndrome (ACS) patients.LL-37 IgG levels were not associated with coronary artery calcium score, but future myocardial infarction patients had significantly higher LL-37 IgG at baseline.Reduced LL-37 IgG in ACS was associated with increased LL-37 IgG-immune complex.ACS plasma increased activated CD62Pþ platelets from healthy donors mediated in part by LL-37 IgG-immune complexes and platelet Fc gamma re- I mmunothrombosis is the interlinked function of innate and adaptive immunity with activation of platelets and the coagulation cascade.It is an aspect of host homeostasis that limits pathogen spread and attempts to localize tissue damage. 1 Unabated inflammation is associated with excessive activation of the immunothrombotic response. 2There is growing consideration that unresolved inflammation is linked to the propagation and prolongation of thrombotic responses such as the one in acute coronary syndrome (ACS). 3,4-37 is the only known cathelicidin antimicrobial peptide in humans and it promotes inflammatory signaling through several mechanisms. 5LL-37 is a component of neutrophil extracellular traps (NETs) that are expelled by degranulating activated neutrophils as part of the innate immune response. 6NETs have been implicated in thrombus formation on culprit plaques in ST-segment elevation myocardial infarction (STEMI). 7Differential gene expression analysis of a cohort of patients with early-onset STEMI under treatment regimen identified increased expression of LL-37 in peripheral blood mononuclear cells (PBMCs), 8 and LL-37 is associated with atherosclerotic cardiovascular disease (ASCVD) risk factors. 9stemic levels of LL-37 are reduced in STEMI patients, even as levels in coronary circulation are increased. 10In addition, a study showed lower systemic LL-37 levels were associated with a higher risk of a recurrent event. 11 previously reported the impairment of T-cell tolerance response to the self-antigen cathelicidin antimicrobial peptide LL-37 in ACS patients. 12The impaired tolerance is characterized specifically by increased CD8þCD69þCD137þ T cells.CD137 mediates the generation of immune memory to antigenic challenges. 13A potential consequence of T-cell responses to LL-37 is the generation of autoantibodies but it is unknown if this occurs in ACS.Furthermore, the role of autoantibodies to LL-37 in ACS remains unknown.
Pathology attributed to LL-37 in autoimmune diseases includes a feed-forward pathway of inflammatory signaling wherein pre-existing LL-37 autoantibodies form immune complexes (ICs) with LL-37 discharged by activated neutrophils as components of NETs. 6,14These LL-37 immunoglobulin G-immune complexes (LL-37 IgG-ICs) then bind Fc gamma receptor 2a (FcgRIIa) expressed by platelets and neutrophils, which then promotes and amplifies further inflammatory responses. 15As such, increased LL-37 [18][19][20] LL-37 IgG levels in ACS have yet to be investigated.
In this report, we evaluated LL-37 IgG levels in the plasma of ASCVD patients in the context of coronary disease severity assessed using the coronary artery calcium score (CACS).Atherosclerotic plaque rupture or erosion are the main causes of ACS and the CACS is a marker of global coronary atherosclerotic burden and a validated risk prediction tool. 21,22The plasma levels of LL-37 IgG in a subcohort of patients in the    C-REACTIVE PROTEIN.C-reactive protein (CRP) levels in EISNER study plasma samples were derived from the existing EISNER database measured using a Luminex Sandwich assay (Alere). 24ACS plasma was measured with CRP Quantikine enzyme-linked immunosorbent assay (R&D Systems) as recommended by the manufacturer.
HEALTHY DONOR PLATELETS.Remnant blood samples contained in leukocyte reduction system cones from platelet donors were acquired from the Cedars-Sinai Medical Center blood bank and subjected to PBMC isolation using a modified Ficoll technique as described. 25Briefly, blood collected in the leukocyte  The relative levels of LL-37 IgG in plasma samples of a subcohort of subjects from the EISNER study 23 were determined.Characteristics of study subjects as grouped are depicted in Table 1.LL-37 IgG levels were compared between patients who had no MI and those who had a future MI.Patients who had a future MI had significantly higher LL-37 IgG levels compared with those who had no MI (Figure 1A).LL-37 IgG levels grouped according to low risk (CACS 0-<100) or high risk (CACS $100) 24 were not different in No MI (Figure 1B) or Future MI (Figure 1C) groups.The results suggest that LL-37 IgG does not segregate according to CACS.To evaluate the inflammatory status of the patients, CRP levels were compared among the lowand high-risk CACS, and Future MI.CRP levels were significantly higher in CACS $100 compared with CACS 0-<100 (Figure 1D).Future MI CRP levels were also elevated but did not reach statistical significance.
The results suggested that LL-37 IgG may be associated less with the chronic inflammatory state and more with the acute event.We then evaluated LL-37 IgG in ACS patients and compared it with Future MI patients.There was significantly less LL-37 IgG in ACS patients compared with Future MI (Figure 1E).No significant differences between female and male LL-37 IgG levels were noted among the different patient groups (Figure 1F to 1I) but the low number of females particularly in the Future MI group limits interpretation of this finding.We then considered the possibilities that either LL-37 IgG levels normalized in the acute patients or LL-37 were bound as LL-37 IgG-ICs given the reported increase in LL-37 levels in coronary circulation in MI patients. 10

LL-37 IgG-ICs ARE INCREASED IN ACS.
There were significantly elevated LL-37 IgG-ICs in ACS patients compared with subjects with CACS ¼ 0 and Future MI patients (Figure 2A).To validate the IgG-IC assay, LL-37 peptide was used to preblock the capture antibody.
There was significant attenuation of the LL-37 IgG-IC signal in ACS plasma with LL-37 peptide preblocking (Figure 2B).The results suggest that although LL-37 IgG levels were less in ACS patients compared with Future MI, this is due in part to increased LL-37 IgG-IC formation.No differences were noted between female and male LL-37 IgG-IC levels in the different patient groups (Figure 2C to 2E).2F).There was no difference in CD154þ platelets with ACS plasma compared with plasma from healthy subjects (Figure 2G).The results support the presence of soluble factors in ACS plasma that increases platelet activation marked by CD62P.

subjects (Figure
To test the role of LL-37 IgG-ICs in ACS patient plasma in platelet activation, LL-37 IgG-ICs were immunedepleted from ACS plasma.

DEPLETION OF LL-37 IgG-ICs REDUCED ACS
PLASMA ACTIVATION OF PLATELETS.LL-37 IgG-ICs in ACS plasma were depleted using sheep anti-LL-37 antibody conjugated magnetic beads.Nonspecific IgG-ICs binding to the antibody host IgG was controlled for using sheep IgG conjugated to magnetic beads.Immune depletion resulted in a modest but significant reduction of ACS plasma LL-37 IgG-IC (Figure 3A).Importantly, immune depletion of ACS plasma LL-37 IgG-ICs had a significant 3-fold inhibition of CD62Pþ platelets compared with 2-fold inhibition by control IgG (Figure 3B).There was no difference in CD154þ platelets by immune depletion of ACS plasma LL-37 IgG-ICs compared with IgG (Figure 3C).

DISCUSSION
The current study extends our previous report on the potential role of self-reactive immune response to the cathelicidin antimicrobial peptide LL-37 in ACS. 12 The study demonstrates that: 1) LL-37 IgG is significantly elevated in intermediate-risk patients who have a future MI; 2) ACS patients have increased LL-37 IgG-ICs; and 3) LL-37 IgG-ICs in ACS patients activates platelets from healthy donors.The combined results support a detrimental role for the adaptive immune response to LL-37 in ACS.
The LL-37 IgG autoantibody levels in plasma samples of study subjects was not related to the extent of ASCVD but instead was significantly higher in patients who had a future MI, suggesting that high LL-37 IgG levels may increase risk for a future acute ASCVD event.Furthermore, we noted the reduced LL-37 IgG autoantibody levels in ACS patients, which was associated with increased detection of LL-37 IgG-ICs, suggesting that the formation of antigen-specific antibody-immune complexes may be functionally important in ACS.This is supported by our findings that LL-37 IgG-ICs promote the activation of platelets from healthy donors marked by increased CD62Pþ platelets.Depletion of the LL-37 IgG-ICs resulted in reduced platelet activation.Our results further support a role for FcɣRIIa in platelet activation induced by ACS plasma demonstrated by the FcɣRIIa blocking experiment.FcɣRIIa avidly binds to IgG-ICs as compared with low affinity for monomeric IgG. 2,26ven that platelets are the most abundant carriers of FcɣRIIa in the blood, it is a key element in IgG-ICinduced platelet activation. 2 The results suggest that LL-37 IgG form ICs with LL-37 in the acute stage and activate platelets mediated in part by FcɣRIIa, which potentiates thrombosis.Taken together, our report supports a potentially detrimental role of LL-37 IgG autoantibodies in ASCVD by increasing the risk for ACS.This is consistent with the report that FcɣRIIa binding to IgG-ICs increases platelet activation and promotes thrombosis in systemic lupus erythematosus. 15The role of LL-37 IgG-ICs specifically in adaptive immunothrombosis is yet to be reported but has been demonstrated to promote pathogenic bone resorption in RA. 27 LL-37 is a component of NETs and itself promotes platelet activation and augments thrombus formation. 28,29Our report adds another layer to the detrimental role of LL-37 in thrombosis by defining a role in the immunothrombotic response in ACS mediated by LL-37 autoantibodies.The combined studies suggest that several properties of LL-37 conspire to bypass endogenous mechanisms of thrombus resolution 30 and lead to the propagation of pathologic thrombosis in ACS.
2][33][34] Importantly, the link between autoimmune/autoinflammatory diseases such as psoriasis and LL-37 was highlighted in a recent report by Nakamura et al 34 demonstrating that LL-37 binding potentiates uptake of LDL by macrophages thereby altering the transcriptional response and promoting the formation of foam cells, adding further to the inflammatory role of LL-37 specifically in ASCVD.
Structural modeling studies in their report suggest that LL-37 binding results in changes in LDL conformation increasing its size and potentially mediating its increased uptake by classic LDL receptors.Transgenic expression of LL-37 in apoEÀ/À mice increased aortic atherosclerosis further supporting its detrimental role in ASCVD. 34UDY LIMITATIONS.A limitation of the study is the small number of patients included in the analysis.These patients are from two different subcohorts selected based on the occurrence of MI during the follow-up period.In addition, comparison was made between patient samples collected from different studies.Consideration must be made for the possibility of selection bias and other covariates not balanced between the groups.The results will need to be validated with a future randomized trial or with a larger prospective cohort study with proper control of baseline imbalance.Another limitation is the lack of increase in CD154 of healthy donor platelets treated with ACS plasma.CD154 is often associated with platelet activation and thrombosis.It is possible that the in vitro assays we performed did not replicate the complex cascade that occurs in ACS patients, or that LL-37 IgG-ICs are more specific to prime CD62P signaling and that CD154 is activated by a different pathway that was not recapitulated in our experiments.Platelet CD62P is an important element in arterial thrombosis, promoting platelet-leukocyte aggregates, 35 through rapid exposure of tissue factor to monocytes, 36 and is associated with the extent of myocardial injury and rate of 30-day major adverse event in STEMI patients. 37

CONCLUSIONS
ceptor 2a.(JACC Basic Transl Sci 2024;9:877-887) © 2024 The Authors.Published by Elsevier on behalf of the American College of Cardiology Foundation.This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

night at 4 C
to assess antibody levels using standard protocol.The coating concentration and serum dilution was optimized in pilot experiments.Horseradish peroxidase anti-human IgG (Southern Biotech) was used to detect antibodies and color development with 2,2 0 -azino-di-(3-ethylbenzthiazoline sulfonic acid) (Southern Biotech) as a substrate.Optical density values were recorded at 405 nm.Pooled blood type A and B antigen (AB) plasma (Innovative Research) was used as a calibrator for the assay and the values expressed as the ratio of each sample to the calibrator.LL-37 IgG-IC ENZYME-LINKED IMMUNOSORBENT ASSAY.Flat-bottomed 96-well polystyrene plates (MaxiSorp) were precoated with 100 mL sheep anti-LL-37 IgG (1 mg/mL in 1x phosphate-buffered saline [PBS]; R&D Systems) overnight at 4 o C. Plates were washed and blocked with 2% bovine serum albumin in 1x PBS for 1 h at 37 o C. Plates were washed and ACS plasma samples diluted 1:100 in 1x PBS were added and incubated for 1 h in 37 o C. Plates were washed and horseradish peroxidase anti-human IgG (Southern Biotech) were used to detect antibodies with 2,2 0azino-di-(3-ethylbenzthiazoline sulfonic acid) (Southern Biotech) as substrate color development.Optical density values were recorded at 405 nm.AB plasma pooled from healthy subjects (Innovative Research) was used as calibrator for the assay and the values expressed as the ratio of each sample to the calibrator.
reduction system cones was reconstituted with an equal portion of PBS containing 2% pooled AB human serum and layered over SepMate Ficoll tubes (Stemcell Technologies).After centrifugation, the layer containing PBMCs and platelets was diluted into 50 mL PBS/2% pooled AB human serum and centrifuged at 350g for 10 min without brakes.The supernatant containing the platelets was collected and further centrifuged at 700g for 10 min without brakes to pellet the platelets.The platelet pellets were resuspended in 1 mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer, counted, and used immediately for experiments.ACS PLASMA ACTIVATION OF HEALTHY DONOR PLATELETS.Four ACS plasma samples that had the highest levels of LL-37 IgG-ICs were selected for platelet activation experiments.Individual plasma samples were diluted to 10% in 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid buffer and a fluorescent antibody mix consisting of CD41, CD61, CD62P, and CD154 was added.Platelets isolated from a single donor were then incubated with individual plasma from the ACS patients in duplicates, with the final plasma concentration at 5%.AB plasma pooled from healthy donors incubated with the platelets also at a final concentration of 5% was used as control.The platelets were incubated in the dark at room temperature for 30 min 15 then 4% paraformaldehyde was added to a final concentration of 1%.The platelets were then pelleted by a 10-min centrifugation at 700g, resuspended in fluorescence-activated cell sorting buffer, and analyzed using flow cytometry.Data are presented as percentage of CD41þCD61þ gated platelets The experiment was repeated with 3 different healthy donor platelets.IMMUNE-DEPLETION OF LL-37 IgG-ICs IN ACS PLASMA.Sheep anti-LL-37 IgG antibody was conjugated to magnetic beads using the Dynabeads Antibody Coupling Kit (Thermo Fisher) according to manufacturer's protocol.Sheep IgG conjugated to magnetic beads was used as control.Anti-LL-37 beads or IgG beads were then used to immune-deplete the 4 ACS plasma samples with the highest levels of LL-37 IgG-ICs overnight at 4 o C. Samples were then subjected to magnetic bead separation and the supernatant collected for platelet activation assays as described already in this article.Each experiment included no magnetic bead incubation corresponding to each plasma sample used.Results are expressed as fold-change relative to no magnetic bead incubation.The experiment was repeated with 2 different healthy donor platelets.BLOCKING OF FcɣRIIa IN PLATELETS.Mouse antihuman FcɣRIIa (clone IV.3, Bioxcell) was preincubated (10 mg/mL) with healthy donor platelets for 15 min at room temperature before addition to individual plasma from the 4 ACS patients with the highest LL-37 IgG-ICs for a final plasma concentration of 5% as described already in this article.Control was no antibody blocking.Plasma samples were premixed with fluorescent antibodies to CD41, CD61, CD62P, and CD154.FcɣRIIa-blocked or control platelets were then added to the plasma samples and incubated in the dark at room temperature for 30 min 15 then 4% paraformaldehyde was added to a final concentration of 1%.The platelets were then pelleted by a 10-min

FIGURE 2
FIGURE 2 LL-37 IgG IC and Platelet Activation

FcɣRIIa
BLOCKING REDUCED ACS PLASMA ACTIVATION OF PLATELETS.FcɣRIIa expressed by platelets is one potential mechanism for LL-37 IgG-ICs to activate platelets.A mouse anti-human FcɣRIIa monoclonal antibody (monoclonal antibody clone IV.3) preincubated with healthy donor platelets was used to evaluate the role of FcɣRIIa in activation of healthy donor platelets by ACS plasma.Individual plasma from the 4 ACS patients with the highest LL-37 IgG-ICs were used to ensure healthy donor platelet activation.Preincubation with FcɣRIIa monoclonal antibody significantly reduced CD62Pþ platelets (Figure 3D).The results support a role for FcɣRIIa in mediating LL-37 IgG-IC activation of platelets through increased CD62P and provide further evidence of the potentially detrimental role of LL-37 IgG-ICs in ACS.

FIGURE 3
FIGURE 3 Immune Depletion of LL-37 IgG-IC and FCɣRIIa Blocking Our study demonstrates a novel pathway of platelet activation in ACS mediated by LL-37 IgG autoantibodies through the formation of ICs potentially promoting excessive inflammatory immunothrombosis.Neutrophil activation during immune and/or traumatic challenges, including coronary atherosclerotic plaque erosion or rupture, leads to NET expulsion of LL-37 autoantigens.The increased levels of LL-37 IgG predisposes the generation of LL-37 IgG-ICs that bind to platelets through FcɣRIIa, resulting in platelet activation and promotion of excessive thrombus formation.FUNDING SUPPORT AND AUTHOR DISCLOSURES 17. Lande R, Palazzo R, Hammel P, et al.Generation of monoclonal antibodies specific for native LL37 and citrullinated LL37 that discriminate the two LL37 forms in the skin and circulation of cutaneous/systemic lupus erythematosus and rheumatoid arthritis patients.Antibodies (Basel).2020;9.https://doi.org/10.3390/antib902001418. Yuan Y, Qiu J, Lin ZT, et al.Identification of novel autoantibodies associated with psoriatic arthritis.Arthritis Rheumatol.2019;71:941-951.19.Moreno-Angarita A, Aragón CC, Tobón GJ.Cathelicidin LL-37: a new important molecule in the pathophysiology of systemic lupus PERSPECTIVES COMPETENCY IN MEDICAL KNOWLEDGE: The report defines a mechanism for platelet activation induced by an autoantibody response specific for a self-antigen in NETs that may contribute to thrombosis in ACS.Autoantibody levels may be clinically relevant in risk assessment of ASCVD patients.TRANSLATIONAL OUTLOOK: Platelet activation and thrombosis are key elements of the attendant risk of ACS in ASCVD patients.Antiplatelet therapy can be enhanced by a clearer understanding of the factors involved in platelet activation and thrombosis mediated specifically by autoantigens in NETs and autoantibody-immune complexes.

TABLE 1
No MI and Future MI Patient Characteristics Values are as mean AE SD, or n (%) and statistical testing with chi-square test and statistical testing with t test or Mann-Whitney test.ACE ¼ angiotensin-converting enzyme; ARB ¼ angiotensin receptor block; BMI ¼ body mass index; DBP ¼ diastolic blood pressure; HDL ¼ high-density lipoprotein; LDL ¼ low-density lipoprotein; MI ¼ myocardial infarction; SBP ¼ systolic blood pressure.

TABLE 2
No MI and Future MI Patient Characteristics Subgrouped Using CACS Values are as mean AE SD, or n (%). a 1 future MI patient did not have CACS on record.CACS ¼ coronary artery calcium score; other abbreviations as in Table1.RESULTSLL-37 IgG IS ELEVATED IN PATIENTS WITH FUTUREMI.